11. Add 50 μL per well of RPMI-FBS containing 50 μg/mL
amikacin.
12. Incubate plates for 1 h at 37
C with 5% CO 2 .
13. Repeat twice steps 9 and 10 with RPMI-FBS.
14. Add 25 μL per well of RPMI-FBS containing 1 μM LysoTracker Red DND-99 or Green DND-26 (see Note 13).
15. Incubate plates for 1.5 h at 37
C with 5% CO 2 .
16. Repeat twice steps 9 and 10 with DPBS-FBS.
17. Add 50 μL per well of a nucleus staining/cell fixative solution
by diluting 10 μg/mL Hoechst 33342 solution in 4% formaldehyde solution.
18. Incubate plates for 30 min at room temperature.
19. Repeat twice steps 9 and 10 with DPBS-FBS.
20. Proceed to image the cells in 50 μL per well of DPBS-FBS,
using the high-content confocal microscope (see Subheading
3.25). Use 60Â magnification. Plates can be stored for up to
1 month, sealed and kept in the dark at 4
C.
3.19 Phagosomal
Acidification Assay
Using pHrodo Reagent
1. Seed 2 Â 10
4 RAW264.7 macrophages and differentiated
CD14
+ hPBMCs (see Subheadings 3.2 and 3.10) in 40 μL per
well of RPMI-FBS supplemented or not with 50 ng/mL
hM-CSF in 384-well plates.
2. Incubate plates overnight at 37
C with 5% CO 2 .
3. Pellet 1 Â 10
9 H37Rv-GFP bacteria (see Subheading 3.1 and
Note 18) in a 1.5-mL microtube by centrifugation at 3750 Â g
for 5 min at room temperature.
4. Resuspend in 140 μL of fresh 100 mM sodium bicarbonate
buffer, pH 8.3.
5. For labeling bacteria, add 10 μL of 7.6 mM pHrodo Red
suspension to obtain a 0.5 mM final concentration and mix
by pipetting up and down several times.
6. Incubate microtube for 60 min at room temperature with
shaking (800 rpm), using a mixed and temperature-controlled
microtube incubator, and protect from light.
7. For removing unincorporated dye from the bacteria conjugate,
add 1 mL of DPBS to bacteria conjugate and vortex the samples for 30 s.
8. Centrifuge at 3750 Â g for 5 min at room temperature.
9. Carefully remove the supernatant using a micropipette (see red
supernatant).
10. Resuspend the bacterial pellet in 1 mL of DPBS.
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Nathalie Deboosere et al.
amikacin.
12. Incubate plates for 1 h at 37
C with 5% CO 2 .
13. Repeat twice steps 9 and 10 with RPMI-FBS.
14. Add 25 μL per well of RPMI-FBS containing 1 μM LysoTracker Red DND-99 or Green DND-26 (see Note 13).
15. Incubate plates for 1.5 h at 37
C with 5% CO 2 .
16. Repeat twice steps 9 and 10 with DPBS-FBS.
17. Add 50 μL per well of a nucleus staining/cell fixative solution
by diluting 10 μg/mL Hoechst 33342 solution in 4% formaldehyde solution.
18. Incubate plates for 30 min at room temperature.
19. Repeat twice steps 9 and 10 with DPBS-FBS.
20. Proceed to image the cells in 50 μL per well of DPBS-FBS,
using the high-content confocal microscope (see Subheading
3.25). Use 60Â magnification. Plates can be stored for up to
1 month, sealed and kept in the dark at 4
C.
3.19 Phagosomal
Acidification Assay
Using pHrodo Reagent
1. Seed 2 Â 10
4 RAW264.7 macrophages and differentiated
CD14
+ hPBMCs (see Subheadings 3.2 and 3.10) in 40 μL per
well of RPMI-FBS supplemented or not with 50 ng/mL
hM-CSF in 384-well plates.
2. Incubate plates overnight at 37
C with 5% CO 2 .
3. Pellet 1 Â 10
9 H37Rv-GFP bacteria (see Subheading 3.1 and
Note 18) in a 1.5-mL microtube by centrifugation at 3750 Â g
for 5 min at room temperature.
4. Resuspend in 140 μL of fresh 100 mM sodium bicarbonate
buffer, pH 8.3.
5. For labeling bacteria, add 10 μL of 7.6 mM pHrodo Red
suspension to obtain a 0.5 mM final concentration and mix
by pipetting up and down several times.
6. Incubate microtube for 60 min at room temperature with
shaking (800 rpm), using a mixed and temperature-controlled
microtube incubator, and protect from light.
7. For removing unincorporated dye from the bacteria conjugate,
add 1 mL of DPBS to bacteria conjugate and vortex the samples for 30 s.
8. Centrifuge at 3750 Â g for 5 min at room temperature.
9. Carefully remove the supernatant using a micropipette (see red
supernatant).
10. Resuspend the bacterial pellet in 1 mL of DPBS.
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Nathalie Deboosere et al.